Deregulation of the imprinted DLK1-DIO3 locus ncRNAs is associated with replicative senescence of human adipose-derived stem cells
Background Human adult adipose-derived stem cells (hADSCs) have become the most promising cell source for regenerative medicine. However the prolonged ex vivo expansion periods required to obtain the necessary therapeutic dose promotes progressive senescence, with the concomitant reduction of their therapeutic potential. Aim and scope A better understanding of the determinants of hADSC senescence is needed to improve biosafety while preserving therapeutic efficiency. Here, we investigated the association between deregulation of the imprinted DLK1-DIO3 region and replicative senescence in hADSC cultures. Methods We compared hADSC cultures at short (P S ) and prolonged (P L ) passages, both in standard and low [O 2 ] (21 and 3%, respectively), in relation to replicative senescence. hADSCs were evaluated for expression alterations in the DLK1-DIO3 region on chromosome 14q32, and particularly in its main miRNA cluster. Results Comparison of hADSCs cultured at P L or P S surprisingly showed a quite significant fraction (69%) of upregulated miRNAs in P L cultures mapping to the imprinted 14q32 locus, the largest miRNA cluster described in the genome. In agreement, expression of the lncRNA MEG3 (Maternally Expressed 3; Meg3/Gtl2), cultured at 21 and 3% [O 2 ], was also significantly higher in P L than in P S passages. During hADSC replicative senescence the AcK16H4 activating mark was found to be significantly associated with the deregulation of the entire DLK1-DIO3 locus, with a secondary regulatory role for the methylation of DMR regions. Conclusion A direct relationship between DLK1-DIO3 deregulation and replicative senescence of hADSCs is reported, involving upregulation of a very significant fraction of its largest miRNA cluster (14q32.31), paralleled by the progressive overexpression of the lncRNA MEG3, which plays a central role in the regulation of Dlk1/Dio3 activation status in mice. ; This work was supported by grants to AB from the Spanish Ministry of Economy, Industry (SAF2015-70882-R; AEI/FEDER, UE), Comunidad Autónoma de Madrid (S2010/BMD-2420), Instituto Salud Carlos III (RETICS TerCel, RD12/0019/0018) and the European Commission (FP7-HEALTH- 2009/CARE-MI). AMS was supported by grants from the MINECO (SAF2010–17167) and Instituto Salud Carlos III (RETICS TerCel, RD12/0019/0013), and MFF and RGU by grants from the Plan Nacional de I+D+I 2013-2016/FEDER (PI15/ 00892), the Asturias Regional Government (GRUPIN14-052), the IUOPA (Obra Social Cajastur) and the Fundación Científica de la AECC. SGL held a predoctoral fellowship from the Spanish Programa de Formación del Profesorado Universitario